Background. The CDC category A bioterrorism agent, Clostridium botulinum neurotoxin (BoNT), is a zinc protease that cleaves proteins involved in presynaptic acetylcholine release thereby causing paralysis. The proteolytically active light chain of BoNT serotype A (BoNT/A) hydrolyzes SNAP-25. Paralysis caused by BoNT serotype A (BoNT/A) can last several months. Any effective treatment must not only gain entry to the affected neurons and inactivate toxin, but must address the fact that BoNT/A has an extremely long duration of action. Hypothesis. We propose that toxin lacking protease activity (iBoNT/A) will be an effective antidote to acute botulism because it should compete for the same substrate as BoNT/A (SNAP-25) without cleaving it and should have the same longevity as BoNT/A. iBoNT/A should enter neurons, localize to same structure on the cell membrane that appears to stabilize the light chain to give its extraordinary long duration of activity, and at high enough intracellular concentrations, displace active light chain, thereby promoting its degradation and re- constituting exocytosis. Preliminary Studies. In recent experiments, we have demonstrated that recombinant BoNT/A light chain inactivated by the introduction of three mutations in the zinc coordination region (iBoNT/A-L) inhibits cleavage of SNAP-25 by native BoNT/A light chain. Specific Aims. We propose to determine whether iBoNT/A can 1) block BoNT/A protease activity in neuronal cells, 2) shorten the duration of action of BoNT/A , and 3) attenuate effects of BoNT/A in mice. Work Proposed. We will determine the inhibition kinetics of native BoNT/A-L activity by iBoNT/A-L and if SNAP-25 cleavage can be inhibited by addition of full length, nicked iBoNT/A to the culture medium of PC-12 cells transfected with BoNT/A-L. We will determine whether iBoNT/A fused to fluorescent protein localizes to PC-12 plasma membrane and colocalizes with SNAP-25 using confocal microscopy. We will examine whether iBoNT/A can inhibit SNAP-25 cleavage in cultured motor neurons given native BoNT/A. The duration of inhibi- tion in these non-dividing cells will be determined. We will determine whether iBoNT/A can attenuate lethality or paralysis by native botulinum serotype A in mice. PUBLIC HEALTH RELEVANCE: Clostridium botulinum neurotoxin (BoNT) is a CDC category A bioterrorism agent. A patient with BoNT sero- type A (BoNT/A) intoxication may spend several months on a ventilator due to respiratory muscle paralysis. Release of a single gram of BoNT could affect several thousand people, thereby crippling local ICUs. There is currently no treatment available that reverses BoNT-induced paralysis. The ultimate goal of these studies is to generate an effective antidote to BoNT/A intoxication.